Resistance toward apoptosis, or programmed cell death, is a hallmark of most, perhaps all, types of human cancer. Bcl-2 proteins are the major regulators of apoptotic signaling pathways, and each Bcl-2 protein member has at least one Bcl-2 homology (BH) domain. Bcl-2 proteins can be classified into anti-apoptotic and pro-apoptotic groups. There are also two classes of pro-apoptotic Bcl-2 proteins: multiple-BH domain and BH3-only. Anti-apoptotic Bcl-2 proteins are believed to protect against disintegration of the mitochondrial outer membrane (MOM) during apoptosis, whereas pro-apoptotic Bcl-2 members promote MOM permeabilization. The expression of individual Bcl-2 protein in different types of cancer has been used as an independent prognostic marker with limited success. Recently profiling mitochondrial sensitivity to a panel of BH3 domains derived from BH3-only Bcl-2 proteins has been shown to more effectively determine the potential of different cancer cells committing apoptosis.
Neoplastic cells often show an increased ratio of anti-apoptotic to pro-apoptotic Bcl-2 proteins, which enables them to survive under adverse conditions. Thus, restoring the aberrant apoptotic pathways in tumor cells might render them more susceptible to stress conditions and subsequent apoptosis. An emerging approach for cancer therapy is to activate the apoptotic pathway directly by reducing the activity of anti-apoptotic Bcl-2 proteins or enhancing the function of pro-apoptotic Bcl-2 proteins. One strategy is to antagonize the anti-apoptotic Bcl-2 proteins. The knowledge about the structures of anti-apoptotic Bcl-2 proteins and their complexes with BH3 peptides have guided the development of small molecules and stapled peptides that indirectly activate the mitochondrial apoptotic pathway by targeting the hydrophobic groove of anti-apoptotic Bcl-2 proteins.
Another less explored approach is to identify small molecules that activate pro-apoptotic Bcl-2 proteins. The activities of the multiple-BH3 Bcl-2 proteins Bax and Bak are redundant, and it is believed that activation of either of them could induce apoptosis in almost all apoptosis paradigms examined. In the majority of cancer cells, Bax protein is functional, but its activities are largely neutralized by often overexpressed anti-apoptotic Bcl-2 proteins. Thus, activation of Bax in tumor cells could be an effective therapeutic strategy. Structural studies have demonstrated that Bax normally resides in the cytosol of healthy cells in an inactive state. The carboxyl-terminal α-helix of Bax is the membrane anchoring region, which is normally sequestered in an inhibitory hydrophobic groove of Bax, preventing its insertion into the MOM. Upon exposure to various death stimuli, through still unknown mechanisms, Bax conformation is changed, and its membrane anchoring domain is exposed and inserted in the MOM. Once translocated into mitochondria, Bax proteins are believed to oligomerize, leading to permeabilization of the MOM and subsequent release of cytochrome c from mitochondria.
In support of this model, in vitro studies using purified mitochondria or reconstituted liposomal systems with BH3 peptides or BH3-only proteins suggest that certain BH3-only proteins, particularly Bid and Bim, can bind to Bax and induce its activation. In addition, biochemical studies also demonstrate that activator BH3-only proteins can bind to the Bax canonical hydrophobic groove to induce Bax oligomerization and activation. Furthermore, crosslinking studies suggest that homo-oligomerization of Bax through an interaction between the BH3 domain and the hydrophobic binding groove (mainly α3-α5) forms “BH3-in-groove.” A recent structural study reveals new detailed information about how certain BH3-only proteins can directly activate Bax, in which BH3 peptides derived from pro-apoptotic Bcl-2 proteins insert into the Bax hydrophobic groove, releasing the core domain (α1-α5) from the latch domain (α6-α8), dislodging the Bax BH3 domain, and subsequently inducing MOM permeabilization.
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